Abstract

Plant biologists have long been studying phenotypic and physiological variation and the molecular mechanisms underlying natural variation in these processes have stimulated scientists to uncover the genomic polymorphism responsible for such variation. Populus tremula, (European aspen, hereafter referred to as aspen), is a member of Populus genus, which has become a model system for genetic and genomic studies and, more recently, for studies linking genomics to ecology and evolution. Many Populus species can be efficiently genetically transformed, all have relatively small genomes of ~500 Mbp and a number of genetic maps have been constructed using various F1 and more advanced crossing designs. Importantly, there is also a reference genome sequence available for P.trichocarpa (Tuskan et al., 2006), which has significantly advanced the utility of Populus as a model system (Wullschleger et al., 2012).Next generation sequencing (NGS) techniques have made genomic studies significantly faster and easier to conduct due to their massively parallel and rapid generation of high-quality sequence data at relatively low cost per base pair. As a result NGS has rapidly become the technology of choice for most scientist conducting sequence projects. NGS has also revolutionized the field of gene expression analysis by its application to sequencing cDNA to assay gene expression levels and is increasingly replacing the use of expression microarrays in genomics studies. This study compared the suitability of two methods for identification of polymorphism that could be used for constructing a genetic map to complement the aspen genome project by facilitating orientation and location of assembly scaffolds within the reference P. trichocarpa genome sequence (Tuskan et al., 2006). We first assessed the applicability of utilizing a reduced representation sequencing library approach in the parents of an F1 intraspecific P. tremula population (RRL). RRL construction involves the use of restriction enzyme digestion of genomic DNA to generate a consistent set of DNA fragment from different samples and serves the purpose of efficiently reducing genomic representation and subsequent volume of sequence data that is required for polymorphism identification.

The second focus of this study was to assess the use of existing gene expression data that had been generated using RNA-Sequencing (RNA-Seq) to generate a de novo reference transcript assembly for one of the parents of the P. tremula F1 population. This reference assembly was then used for Single Nucleotide Polymorphism (SNP) detection using RNA-Seq data from both parents to identify a set of SNPs would then be suitable for genotyping the F1 progeny to allow construction of a genetic map.

Main title:

A next generation sequencing approach to constructing a genetic map for Populus tremula